Preliminary Study Of Gene Expression Signatures In Hereditary Nonpolyposis Colorectal Cancer

Hereditary nonpolyposis colorectal cancer(HNPCC) is one of the most common autosomal dominantly inherited cancers syndrome, accounts for 2%-15% of all colorectal cancers and the penetrance reach up to 80%-90%. Compare with sporadic colorectal cancer(SCRC), HNPCC shows its own characteristcs associated with the molecular mechanism,clinical features,the methods for treatment and management of HNPCC kindreds. Therefore, to differentiate the HNPCC from SCRC is very important and it will interest not only in the clinic but also in genetic counseling of HNPCC kindreds. Now, many countries and territories have established the clinical diagnostic criteria for HNPCC, such as Amsterdam Criteria and Bethesda Guidelines. The molecular genetics basis of HNPCC was the mutation of mismatch repair gene, which can induce the increasement of misreplications, microsatellites instability and then result in tumorigenesis of many organs.Now, human MMR gene which has been locolized and cloned as hMLH1,hMSH2,hMSH6,hMSH3,hPMS1,hPMS2 and so on were closely associated with HNPCC. The former two account for a large majority of mutations found in HNPCC families from various countries. Previously, We detected germline mutations and large genomic variantions of the entire coding regions of hMSH2lhMLH1/hMSH6 genes and the methylation of hMLHl promoter in 58 HNPCC families fulfilling different cliinical criteria, and the final germline-variation rate of Chinese HNPCC was 53.4%(31/58). It has been reported that the germline mutation of hMSH2, hMLHl and hMSH6 was about 42%-75% in HNPCC families fulfilling different clinical criteria, while the mutation rate of MMR gene in the atypical HNPCC family was likely to less. Some HNPCC families which can found the mutation of MMR gene didn't fulfil any clinical criteria, one the other hand, more than fifty percent suspected HNPCC patients can't found mutation of MMR gene. Therefore, the tumorigenesis of HNPCC must be associated with other disease genes which has not been verified. With the development of gene chip technic, people can study the gene expression profile on the level of genomewide, and recognize the relationship of the gene expreesion and tumorigenesis, progress and metastasis on a whole level. Many related reseach has been reported on the gene expression profile of breast cancer, lung cancer, protaste cancer, lymphoma SCRC and so on. But the study on the HNPCC gene expression has not been reported up till now.In this study, we collected 50 HNPCC families fulfilling different clinical criteria, detected the germline mutation and protein expreesion of MLH1 and MSH2, then we devided the 50 samples into two groups:MMR-proficient and MMR-deficient phenome. Secondly, we use the the Human Genome U133A GeneChip array (Affymetrix) to analysis the the gene expreesion of this 50 samples and another 7 SCRC samples. Thirdly, we screen the differential expression gene of the HNPCC and SCRC, MMR-proficient and MMR-deficient phenome, constructed the gene signature of HNPCC and MMR-deficient tumors. Finally, we demonstrated the robustness of the signature by transferring it to a real-time RT-PCR and IHC platform.This two-step classification approach can identify MMR-deficient phenome as well as HNPCC cases merits further gene expression studies to identify prognostic signatures.The current research project is comprised for the following three parts:Object:To analyse the expression of hMLH1 and hMSH2 protein in 50 HNPCC probands fulfilling defferent clinical criteria and the germline mutation of hMLHl and hMSH2 gene in 26 probands.Methods:The peripheral blood was collected from the probands of 26 HNPCC families fulfilling different clinical criterial, of which 7 families fulfilled AC,19 kindreds fit BG. Genomic DNA was extracted following the manafacturer's protocol and used as the template to amplify 35 exons of the 2 genes by PCR. PCR products were purified and used as the template for direct DNA sequencing. The results of sequencing were analysed by different bioanalysis software. To further investigate the pathological effects of detected missense mutations, we analyse the same exon of hPMS2 gene using PCR-based sequencing in 130 healthy persons without family history. IHC envision two step method was performed.Results:8 germline mutation of hMLHl and hMSH2 was found in 26 HNPCC probands families.4 fulfilled AC and 4 fulfilled BG.17 probands was found to be hMLHl and hMSH2 protein deficient.Conclusions:The rate of germline mutation and protein expression was 30.8% and 34%. AC are the most sensitve clinical creteria to predict mutations. Immunohistochemical staining are reliable screening method with high predictive value for the detection of mutation. Some suspected HNPCC patients can't found mutation of MMR gene.Object:To screen the differential genes between HNPCC and SCRC, MMR-proficient and MMR-deficient phenotype.Methods:Collected the 50 HNPCC probands and 7 SCRC tumor specimen. Labelling of RNA, hybridisation and scanning. Biotin-labelled cRNA was prepared from 10μg of total RNA and hybridised to the Human Genome U133A GeneChip array (Affymetrix). The readings from the quantitative scanning were analysed by SAM and the CapitalBio(?) Molecule Annotation System V4.0.Results:Results in 425 differential expression genes btween HNPCC and SCRC,445 genes between AC and SCRC,469 between FD and SCRC,153 between BG1 and SCRC,613 between RB and SCRC,662 between RB and SCRC for further analysis.Conclusions:58 gene was found for further analysis and to accompany the differential genes between HNPCC and SCRC besides MMR gene. A eleven-gene signature was constructed to seperating MMR-proficient and MMR-deficient phenotype.Object:To demonstrate the differential expression genes between HNPCC and SCRC, screen and preliminary identify molecular signature of MMR-deficient phenotype in 112 cases(included the 57 cases in part 2).Methods:Tissue section from 28 HNPCC and 28 SCRC patients were examined using real-time quantitative reverse transcription polymerase chain reaction(RQ-RT-PCR) technique for ANPEP, MTA2, APCDD1 and HEPACAM, and immunohistochemistry for the expression of the former three genes. Another 56 cases of HNPCC and SCRC were also used to perform the former three genes immunostaining. Demonstrate the exression of the four genes in different groups.Results:The expression of ANPEP, MTA2 in HNPCC was significantly higher than in SCRC, while APCDD1, HEPACAM was lower in HNPCC. The results was coincidence with the gene chip. The expression of these four genes was also different between the group of MMR-proficient and MMR-deficient phenotype.Conclusions:ANPEP, MTA2, APCDD1 and HEPACAM may be a signature capable of separating HNPCC and SCRC as well as MMR-proficient and MMR-deficient phenotype.